Characterization of Meloidogyne indica (Nematoda: Meloidogynidae) Parasitizing Neem in India, with a Molecular Phylogeny of the Species

Tender neem plants showing the symptoms of stunting and yellowing were collected along with the rhizosphere soil from Jaipur district, Rajasthan, India; located at N26°55′10″ and E75°47′16″. The soil type was found to be sandy to sandy loam with medium phosphorus and medium to high potassium content. Average annual rainfall in the area ranges from 500 to 600 mm (Anonymous, 1995). For diagnosis and identification purposes, females were directly dissected out of the galled roots for preparing perineal patterns and genomic DNA extraction. Eggmasses were hand-picked from the infected neem roots and were used for hatching the second-stage infective juveniles. Both second-stage juveniles (J2s) and males were collected by the modified Baermann’s technique (Whitehead and Hemming, 1965).

For conducting taxonomic studies, the J2s and males were processed by the glycerol–ethanol method following standard procedures (Seinhorst, 1959). The morphological characters used for taxonomic identification were employed as described by Karssen (2002) and Carneiro et al. (2014). Morphometric illustrations and measurements (in μm) were made with a camera lucida and an ocular micrometer within an Olympus BX50 compound microscope. The posterior portion with cuticular markings surrounding the vulva and anus of stained-fixed females was cut using a fine-pointed scalpel to obtain the perineal pattern. The inner tissue was carefully removed using flexible bristle, trimmed and transferred to a drop of anhydrous glycerol on a clean glass slide (Southey, 1986) to study the perineal pattern morphology. Photomicrographs of perineal pattern, females, males, and J2s were taken with a Zeiss Axiocam M2m compound microscope equipped with differential interference contrast optics.

Scanning electron microscopy (SEM) was done using a JEOL (UK) JSM 6700 FEG scanning electron microscope equipped with a GATAN (UK) Alto 2500 Cryo unit. The instrument was prepared by cooling with liquid nitrogen and the temperature in the microscope chamber was maintained at −160°C throughout sample examination. The nematode specimens were pipetted onto a 595-grade Whatman^TM filter paper attached to a cryo stub with OCT mountant (Sakura Finetek, Europe NL) and frozen by plunging into pre-slushed liquid nitrogen. The sample was then transferred to the GATAN cryo chamber stage under vacuum and etched to remove contaminating ice by increasing the temperature to −95°C for 1 min. Once the temperature of the stage had returned to −160°C, the sample was coated with Au/Pd alloy for 1 min and transferred to the SEM chamber and mounted on the microscope stage for examination at −160°C. Images were recorded using the JEOL on board system and software.

For molecular analysis, single adult female was lysed in worm lysis buffer (Subbotin et al., 2000) and the molecular markers, viz., ITS, D2D3, and COI were PCR amplified using the primers listed in Table 1. The reaction condition for amplification of the ITS gene was followed as described in Subbotin et al. (2001, 2006). For D2D3 and COI genes, the thermal cycling program was used as described in Ye et al. (2007). Amplified products were separated by electrophoresis on 1% agarose gel and purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The gel purified PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α competent cells (New England Biolabs). Recombinant plasmids were isolated from the positive clones (QIAGEN Plasmid Miniprep kit (Qiagen, Valencia, CA, USA)) and sequenced. The raw sequences were checked for quality using MEGA6 (Tamura et al., 2013), aligned, and a consensus sequence was generated for each gene using Bioedit software (Tom Hall; Ibis Biosciences, Carlsbad, CA).

The obtained ITS, D2D3, and COI sequences were compared with the available sequences in the NCBI GenBank database using BLAST search. Corresponding published sequences (of Meloidogyne spp.) for each gene were retrieved (Table 2) and phylogenetic analyses were carried out using MEGA6 software (Tamura et al., 2013). The sequences were aligned using ClustalW and evolutionary history was deduced by the maximum likelihood (ML) method with selection of appropriate model using Modeltest (Posada and Crandall, 1998). The phylograms were bootstrapped 1,000 times (Felsenstein, 1985) to assess the degree of support for the phylogenetic branching as indicated in the consensus tree. Pratylenchus vulnus was used as out-group.

The sequences were deposited in the NCBI GenBank with the accessions KC311146, MF680038, and MF662179 for ITS, D2D3, and COI, respectively.

Inoculation assay was conducted in a soil system to test whether the nematode species infecting neem can parasitize and multiply on citrus. One-month-old citrus seedlings were planted in plastic pouches (height: 6 inch, diameter: 4 inch) filled with sterilized soil, and inoculated with 2 J2s/g soil. Freshly hatched J2s from the eggmasses, collected from neem roots, were used for inoculation. The experiment was conducted with five replicates.

The infected neem seedlings showed the symptoms of yellowing of leaves coupled with stunted growth of the immature plants. Examination of the infected plants revealed the presence of heavy galling on the roots located mostly along the tap root axis with few lateral branches (Fig. 1A–C). For characterization and identification of the species, morphology of adult females, males and J2s was studied (Fig. 1D–K) including SEM photomicrographs (Fig. 2) with minute detailing.

The nematode species infecting neem was found to infect the citrus plants. The citrus roots showed the presence of galls with formation of eggmasses when observed at 35 days post inoculation (Fig. 3). The females, dissected out from the citrus roots, showed similar perineal pattern morphology with that of the neem population (data not shown).

Fig. 1

Fig. 2

Fig. 3